NZ256425A

NZ256425A - Sec 1 suppressor sso gene and its use in increasing secretion of proteins through over expression especially applicable in biomass production

Info

Publication number: NZ256425A
Application number: NZ256425A
Authority: NZ
Inventors: Sirkka Keranen; Markku Aalto; Mika Outola; Hans Ronne; Merja Penttila
Original assignee: Valtion Teknillinen
Priority date: 1992-10-06
Filing date: 1993-10-06
Publication date: 1996-08-27
Also published as: AU5112793A; WO1994008024A1; ATE254663T1; CA2146240A1; FI924494A0; NO319132B1; EE03074B1; JP3638599B2; EP0665890B1; EP0665890A1; NO951315L; NO951315D0; US5789193A; ES2211870T3; FI114482B; DE69333304D1; DK0665890T3; FI951633A; CA2146240C; AU678402B2

Abstract

(A) An isolated DNA sequence of a sec1 suppressor gene SSO or a homologue is claimed, which DNA sequence, when overexpressed in eukaryotic cells, renders the cells capable of producing increased amts. of secreted proteins. Also claimed are: (B) a vector comprising a DNA sequence as in (A); and (C) recombinant eukaryotic cells carrying a DNA sequence as in (A) and expressing enhanced levels of SSO protein.

Description

<div class="application article clearfix" id="description"> New Zealand No. 256425 International No. PCT/FI93/00402 Complete Specification Filed: Class: (6) S. .Q^.;... P.O. Journal No:... .lifli £< J j i. m at- '•* tow — NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of Invention: Increased production of secreted proteins by recombinant eukaryotic cells Name, address and nationality of applicant(s) as in international application form: VALTION TEKNILLINEN TUTKIMUSKESKUS, of Vuorimiehentie 5, FIN-02150 Espoo, Finland .'sU ca: WO 94/08024 PCT/FI93/00402 ► ■ 256425 INCREASED PRODUCTION OF SECRETED PROTEINS BY RECOMBINANT EUKARYOTIC CELLS Field of the invention 5 This invention relates to recombinant-DNA-technology. Specifically this invention relates to new recombinant eukaryotic cells transformed with SSO genes or their homologues. A eukaryotic cell transformed with several copies of a SSO gene or a gene homologous to SSO has an increased capacity to produce secreted foreign or 10 endogenous proteins. Further, the said new recombinant eukaryotic cells, especially yeasts and filamentous fungi, when transformed with genes expressing suitable hydrolytic enzymes can hydrolyze and/or utilize appropriate macromolecular/polymeric compounds more IS efficiently, which results in increased cell mass production and/or more versatile utilization of the compounds in relevant biotechnical applications. Background of the invention 20 The development of recombinant DNA methods has made it possible to produce proteins in heterologous host systems. This possibility greatly facilitates production of e.g. proteins of therapeutic importance which normally occur in nature in very low amounts or are otherwise difficult to isolate or purify. Such proteins include growth factors, hormones and other biologically active proteins or peptides which 25 traditionally have been isolated from human or animal tissues or body fluids e.g. blood serum or urine. The increasing danger of the presence of human pathogenic viruses such as HBV, HTV, and oncogenic viruses or other pathogens in the human or animal tissues or body fluids has greatly speeded up the search for heterologous production systems for these therapeutics. Other proteins of clinical importance arc 30 viral or other microbial or human parasite proteins needed for diagnostics and for vaccines especially of such organisms which are difficult to grow in vitro or in tissue WO 94/08024 PCT/FI93/00402 ^ 2 culture, or are dangerous human pathogens. These include viruses like HBV, HIV, yellow fever, rubella, FMDV, rabies, and human parasites such as malaria. A further group of proteins for which heterologous production systems have been or 5 are being developed are secreted enzymes, especially those hydrolyzing plant material, and which are needed in food and fodder production as well as in other industrial processes including textile industry and pulp and paper industry. The possibility of producing proteins in heterologous systems or production of endogenous proteins in genetically engineered cells increases their yields and greatly facilitates 10 their purification and has already by now had a great impact on studies of structure and function of many important enzymes and other proteins. The production and secretion of foreign hydrolytic enzymes in yeast for example, results in improvements in processes based on industrial yeast strains such as distiller's, brewer's or baker's yeasts. 15 Various production systems have been and are being developed including bacteria, yeasts, filamentous fungi, animal and plant cell cultures and even multicellular organisms like transgenic animals and plants. All of these different systems have their advantages, even if disadvantages, and all of them «re needed. 20 The yeast Saccharomyces cerevisiae is at the moment the best known eukaryote at genetic level. As a eukaryotic microbe it possesses the advantages of a eukaryotic cell like most if not all of the post-translational modifications of eukaryotes, and as a microbe it shares the easy handling and cultivation properties of bacteria. The large 25 scale fermentation systems are well developed for S. cerevisiae which has a long history as a work horse of biotechnology including production of food ingredients and beverages such as beer and wine. The yeast genetic methods are by far the best developed among eukaryotes based on 30 the vast knowledge obtained by classical genetics. This made it easy to adopt and further develop for yeast the gene technology procedures first described for Escherichia coli. Along other lines the methods for constructing yeast strains 94/08024 PCT/ FI93/00402 3 producing foreign proteins have been developed to a great extent (Roraanos et. al., 1992). Secrction of the proteins into the culture medium involves transfer of the proteins through the various membrane enclosed compartments constituting the secretory pathway. First the proteins are translocated into the lumen of the endoplasmic reticulum ER. From there on the proteins are transported in membrane vesicles to the Golgi complex and from Golgi to plasma membrane. The secretory process involves several steps in which vesicles containing the secreted proteins are pinched off from the donor membrane, targetted to and fused with the acceptor membrane. At each of these steps function of several different proteins are needed. The yeast secretory pathway and a great number of genes involved in it have been elucidated by isolation of conditional lethal mutants deficient in certain steps of the secretory process (Novick et al, 1980; 1981). Mutation in a protein, needed for a particular transfer step results in accumulation of the secreted proteins in the preceding membrane compartment. Thus proteins can accumulate at ER, Golgi or in vesicles between ER and Golgi, or in vesicles between Golgi and plasma membrane. More detailed analysis of the genes and proteins involved in the secretory process has become possible upon cloning the genes and characterization of the function of the corresponding proteins. A picture is emerging which indicates that in all steps several interacting proteins are functioning. We have recently cloned two new yeast genes, SSOl and SS02 as multicopy suppressors of seel -1 defect in growth and secretion in elevated temperatures (Aalto et al., 1993). Many of the genes identified in and isolated from S. cerevisiae have been found and cloned from other organisms based either on the sequence homology with yeast genes or complementation of yeast mutations. Mammalian NSF factor is the homologue of yeast SEC18 gene product and displays a similar function in protein secretion (Wilson et al., 1989). SEC14 gene of Yarrowia lipolytica (Lopez et al, 1992) has been cloned and characterized. Mammalian homologue for yeast SEC11 gene coding for a 94/08024 PCT/FI93/00402 4 component of the signal peptidase has been cloned (Greenberg et al, 1989). Schizosaccharomyces pombe YPT1 gene coding for a small GTP binding protein was cloned using the yeast gene SEC4 as a probe (Fawell et al., 1989) and the mammalian counterpart of YPT1 was shown to be part of the secretory machinery using antibodies against the yeast Yptl protein (Segev et al, 1988). Mammalian rabl protein shown to be homologous to Yptlp (Zaraoui et al, 1989) can substitute for yeast Yptl function (Haubruck et al, 1990). Genes homologous on the protein level to the yeast SSOl and SS02 genes according to the invention are found in several species including mouse (Hirai et al, 1992), rat (Inoue et al, 1992, Bennett et al, 1992) and nematode (Ainscough et al, 1991; EMBL Data Bank 29, accession number M 75825) indicating that the gen^s are conserved during evolution. The homologous proteins in the other species also appear on the cell surface or are implicated to be involved in synaptic vesicle transport to the cell surface, suggesting that they may be functionally related to SSOl and SSO2. However, direct involvement in secretion has only been demonstrated for the Sso-proteins, reported by us (Aalto et al., 1993). Yeast homologues for the synaptic vesicle membrane proteins, synaptobrevins are the Sncl and Snc2 proteins (Gerst et al 1992; Protopopov et al 1993). The above examples, many more of which exist, illustrate the universal nature of the secretory machinery. Results obtained with yeast are largely applicable to other fungi as well as other eukaryotic cells. Genes with sequence similarity to the SSO genes are implicated to function also in other steps of intracellular protein transport/secretion: SED5 (Hardwick and Pelham, 1992) between ER and Golgi and PEP12 (Becherer and Jones, 1992) between Golgi and vacuole, the lysosome compartment of yeast. This further supports the central and conserved role of the SSO genes in protein secretion and intracellular transport. However, no reports exist so far on any positive effect of the &SO-homologues in yeast or animal cells on secretion when overexpressed, which effect we are showing ir this invention for the SSO genes. WO 94/08024 PCT/FI93/00402 5 Less is known about the secretory system of other yeasts such as Kluyveromyces, Pichia, Schizosaccharomyces and Hansenula, which, however, have proven useful hosts for production of foreign proteins (Buckholz and Gleeson, 1991). The genetics and molecular biology of these yeasts arc not as developed as for Saccharomyces but 5 the advantages of these yeasts as production hosts are the same as for Saccharomyces. This holds true also for filamentous fungi such as Neurospora, Aspergillus and Trichoderma which have been used for production of secreted foreign proteins (Jeenes et al., 1991). Belonging taxonomically to Fungi and very many of the filamentous fungi even belonging to Ascomycetes, like S. cerevisiae does, it is evident 10 that the secretory machinery of filamentous fungi is similar to that of S. cerevisiae. Filamentous fungi are very efficient in secreting their own hydrolytic enzymes. However, production of foreign proteins in filamentous fungi is much less efficient and in many cases this seems to be due to inefficient secretion. The common features of all fungi are for instance post-translational modifications occurring along the 15 secretory pathway. Several attempts have been made and published previously to increase foreign protein production in yeast and filamentous fungi as well as in other organisms. Much work has been devoted to various promoter and plasmid constructions to increase the 20 transcription level or plasmid copy number (see e.g. Baldari et al 1987; Martegani et al 1992; Irani and ICiigore, 1988). A common approach to try and increase secretion is to use yeast signal sequences (Baldari, et al. 1987, Vanoni et al 1989). Random mutagenesis and screening for a secreted protein (Smith et al, 1985; Sakai etal., 1988; Schuster et al, 1989; Suzuki et al, 1989; Sleep etal, 1991; Lamsa and 25 Blocbaum, 1990; Dunn-Coleman et al., 1991) or fusion of the foreign protein to an efficiently secreted endogenous protein (Ward et al, 1990; Harkki et al., 1989; Nyyssdnen et al. 1993; Nyyss6nen et al, Pat. Appl.) have been widely used both for yeast and filamentous fungi in order to make the secretion of foreign proteins more efficient. Both of these methods are of limited use. Overproduction mutants isolated 30 by random mutagenesis and screening are almost exclusively recessive and thus cannot be transferred into industrial yeast strains which are polyploid. Often the overproduction results from changes other than increased secretion and in many cases WO 94/08024 PCT/F193/00402 * affects only the protein used for screening. Fusion protein approach requires tailoring of the fusion construction for each foreign protein separately. Our approach, increasing the copy number of genes functioning in secretion and thus 5 the amount of components of the secretory machinery is more universal: it is applicable to any protein without specific fusion constructions and applicable to diploid and polyploid strains. It is not exactly known which steps form the bottle necks in the secretory process, but 10 it can be anticipated that there are several ones of them. We started to unravel the potential blocks at the very end of the secretory pathway, and have cloned and characterized genes participating at the very final stage of the secretory process at which the secretory vesicles budding from the Golgi complex are targetted to and fused with the plasma membrane to release the secreted proteins to the cell exterior. 15 We have previously cloned and characterized SEC1 functioning at this stage (Aalto et al, 1991; Aalto et al, 1992) and have later shown that SEC1 is an essential single copy gene (Aalto et al, 1993). The SSO genes according to the invention were cloned as multicopy suppressors of secl-1 mutation (Aalto et al., 1993). 20 Summary of the invention The present invention describes the isolation of genes which, when overexpressed enhance the production of secreted proteins. Specifically, the present invention describes the isolation of SSOl and SS02 genes of & cerevisiae coding for Ssolp and 25 Sso2p, respectively, the characterization of the genes and their transfer into, and overexpression in S. cerevisiae. In addition, this invention describes isolation of a SSO homologue from Trichoderma reesei, characterization of the gene, and transfer and overexpression in Trichoderma. 30 Furthermore, the sequence homologies between the yeast SSO genes and their higher eukaryotic counterparts indicates that this invention can be used to construct novel cell lines for higher eukaryotes with increased secretion capacity. PCI/ F1 9 3 / 0 0 4 0 2 21 -12- 1994 7 This invention thus provides new recombinant eukaryotic cells, preferably fungal host cells expressing enhanced levels of Sso protein(s), and especially yeast strains expressing enhanced levels of Ssol and/or Sso2 proteins as well as Trichoderma strains expressing enhanced levels of Trichoderma Sso-protein. This invention also provides process(es) for production of increased amounts of secreted proteins by overexpressing genes interacting with the SSO genes, such as SEC1. The eukaryotic cells according to the invention being transformed with the SSO genes or genes interacting with the SSO genes have an increased capacity to produce secreted proteins. The new eukaryotic cells according to the invention, especially yeast and filamentous fungi, can also be used for more efficient production of hydrolytic enzymes and hydrolysis of e.g. polymeric substrates which results in improvements in biotechnical processes such as single cell or baker's yeast production due to increased cell mass or in other processes where efficient production of hydrolytic enzymes and/or efficient hydrolysis of plant material is beneficial. Brief description of the drawings Figs. 1A and IB show the S. cerevisiae SSOl and SS02 gene cDNA integrated into a multicopy plasmid pMAC561 resulting in plasmids YEpSSOl and YEpSS02, respectively. Fig. 2 shows Western analysis demonstrating overexpression of Sso2 protein in yeast transformed with YEpSS02. Fig. 3 shows increased production of secreted Bacillus a-amylase by S. cerevisiae (strain sf750-I4D) transformed with multicopy plasmid, expressing SSOl or SS02 gene and with another plasmid expressing Bacillus a-amylase gene. Fig. 4 shows Western analysis of Bacillus a-amylase secreted by S. cerevisiae with or without the multicopy plasmid expressing SSOl or SS02 gene. .r?px \„/in«-o P J, ; i, ,.,-i WO 94/08024 PCT/FI93/00402 8 Fig. 5 shows increased production of secreted Bacillus a-amylase by S. cerevisiae (strain DBY746) transformed with multicopy plasmid, expressing SS02 gene and with another plasmid expressing Bacillus a-amylase. 5 Fig. 6 shows increased production of secreted Bacillus a-amylase by S. cerevisiae (strain DBY746) transformed with a multicopy plasmid expressing SEC1 gene and with another plasmid expressing Bacillus a-amylase. Fig. 7 shows the SS02 expression cassette flanked by ribosomal sequences integrated 10 into BS+, generating the vector pRbSS02. Fig. 8 shows hybridization of DNA derived from six different fungal species with the yeast SSOl gene. 15 Detailed description of the invention For better understanding of the following detailed description of the invention it may be helpful to give definitions of certain terms to be used hereinafter. 20 Overexpression of a gene: The protein encoded by the said gene is produced in increased amounts in the cell. This can be achieved by increasing the copy number of the gene by introducing extra copics of the gene into the cell on a plasmid or integrated into the genome. Overexpression can also be achieved by placing the gene under a promoter stronger than its own promoter. The amount of the protein in the 25 cell can be varied by varying the copy number of the gene and/or the strength of the promoter used for the expression. Suppression of a mutation: When the effect of a mutation in a given gene is alleviated or abolished by a mutation in an other gene, this second gene is called a 30 suppressor of the first gene. Suppression can occur also by overexpression of the wild type allele of the second gene by the means described above. This" is called overexpression suppression. If the overexpression is caused by multiple copies of the WO 94/08024 PCT/F193/00402 suppressing gene the suppression can also be called multicopy suppression. Suppression phenomenon indicates that these two genes interact at genetic level. The interaction may also occur at physical level as direct, physical contact between the two proteins encoded by the interacting genes. 5 Homologous genes, homologues: Genes which are related, but not identical, in their DNA sequence and/or perform the same function are homologous with each other and are called each other's homologues. 10 Secreted proteins: Proteins which inside of the cell are directed to the secretory pathway and transported through it to the exterior of the cell, outside of the plasma membrane, are called secreted proteins. In yeast the proteins may remain associated with the cell wall such as invertase or released through the cell wall into the growth medium such as the foreign protein Bacillus a-amylase. 15 SSOl and SS02 genes to be used in this invention are isolated from an organism containing these genes e.g. Saccharomyces cerevisiae and Trichoderma spp. Also other suitable yeasts and other fungi, such as Schizosaccharomyces pombe, KLuyveromyces lactis, Pichia spp., Hansenula spp., Aspergillus spp., Neurospora spp. 20 and Penicillium spp. can be used. It is to be noted that homologous genes from other organisms can also be used. Furthermore, overexpression of other genes functioning at the same step with the SSO genes, such as SEC1, in the presence of normal or increased levels of Sso-proteins 25 results in increased secretion. Genes functioning at the preceding steps of the secretory process may well have a similar effect. Thus, release of the secretory vesicles from the Golgi compartment may be facilitated by increasing the copy number of SEC7 and/or SEC14 genes known to function at this step (Novick et al. 1980) or by searching for and increasing the copy number of genes interacting with 30 SEC7 and/or SEC14 e.g. suppressors of their mutations. Likewise any previous step of the secretory process may be improved by increasing the copy number of gches involved. The new genes we have isolated from S. cerevisiae, SSOl and SS02 WO 94/08024 PCT/FI93/00402 10 represent duplicated genes which suggests that they play an important role in the cell. Based on the conserved nature of SSOl and SS02 and their homologues in other species, as mentioned above, we propose that increase of the SSO genes in any other eukaryotic species would result in increased protein secretion efficiency including 5 other yeasts, filamentous fungi, and plant and animal cells. It is to be noticed that due to the fact that many genes involved in secretion function in other organisms, this invention covers for instance also expression of yeast genes in filamentous fungi and higher eukaryotes and vice versa, or any eukaryotic gene in 10 another eukaryote to obtain enhanced secretion. The host to be transformed with the genes of the invention can be any eukaryotic cell suitable for foreign or endogenous protein production, e.g. any 5. cerevisiae yeast strain, (e.g. DBY746, AH22, S150-2B, GPY55-15Ba, VTT-A-63015) any 15 Trichoderma spp. such as T. harzianum and the T. reesei strains derived from the natural isolate QM6a, such as RUTC-30, QM9416 and VTr-D-79125, any JQuyveromyces spp., Sch. pombe, H. polymorpha, Pichia, Aspergillus, Neurospora, Yarrowia, Penicillium spp. or higher eukaryotic cells. Transfer of the genes into these cells can be achieved, for instance, by using the conventional methods described for 20 these organisms. The DNA sequence containing SSOl or SS02 is isolated from S. cerevisiae by conventional methods. In a preferred embodiment gene or cDNA library on a multicopy plasmid is used to suppress the temperature-sensitivity of seel -1 mutant 25 (Aalto et al, 1991; 1993) or mutations leading to deficiency in the SSO function of S. cerevisiae or analogous mutations of other species. In another approach the known DNA sequence of the SSO genes and SSO-like genes is used to design probes for heterologous hybridization or PCR primers for cloning the SSO genes. In still another approach antibodies to the known SSO and £SO-like genes are used for cloning the 30 gene by standard methods. 94/08024 PCT/FI93/00402 11 The genes corresponding to the S. cerevisiae SSOl and SS02 are isolated from the other fungi or higher eukaryotes with one or several of the following methods, which are here described specifically for the filamentous fungus Trichoderma reesei and which can b? modified according to conventional knowledge and means to suit the eukaryotic cell in question. A cDNA bank of T. reesei is constructed into the yeast vector pFL60 as described in the FI patent application No. 92 2373 (Buchert et al.). This gene bank DNA is transformed into the Sxerevisiae strain H4S8 (Aalto et al1993) and screened for complementation of the secretion defect e.g. as described in Example 6. The plasmid is isolated from the positive colonies and the gene is isolated and characterized using standard methodology, and the corresponding chromosomal gene is isolated. Succesful complementation shows that functionally equivalent genes to the yeast SSO genes exist in other fungi such as T. reesei. Alternatively, the genes encoding proteins corresponding to the S cerevisiae Ssolp and/or Sso2p can be isolated from a cDNA or a chromosomal gene bank prepared from T. reesei by heterologous hybridization in non-stringent conditions as described in Example 7 and characterized by conventional methods and their function can be shown as described above. Similar approach is suitable for all organisms which have shown to possess chromosomal sequences homologous to the yeast SSO genes as analyzed for instance by Southern hybridization of total DNA. It is also possible that the gene can be isolated from an expression library with antibodies prepared against the yeast Sso proteins. Alternatively, oligonucleotide primers can be designed based on the homologies found between the sequences of the corresponding genes isolated from several organisms. Clear homologies are seen for instance in regions extending from aa 266 to aa 287 in Ssolp and from aa 269 to aa 290 in Sso2p, shown in SEQ ED NO. 1 and SEQ ID NO. 3, respectively. These primers arc used to amplify the T. reesei gene in a PCR reaction. WO 94/08024 PCT/FI93/00402 To construct a plasmid suitable for transformation into a yeast, the SSOl or SS02 gene is cloned into a suitable yeast expression vector, such as pAAHS (Ammerer, 1983) or vectors derived from it (Ruohonen et al., 1991; Ruohonen et al., manuscript in preparation, a) comprising the appropriate yeast regulatory regions. These 5 regulatory regions can be obtained from yeast genes such as the ADH1, GAL1 -GAL10, PGK1, CUP1, GAP, CYC1, PHOS, or asparagine synthetase gene, for instance. Alternatively, also the regulatory regions of SSOl or SS02 can be used to express the genes in S. cerevisiae. The plasmid carrying the SSOl or SS02 gene is capable of replicating autonomously when transformed into the recipient yeast strain. 10 The gene SSOl or SS02 together with the appropriate yeast regulatory regions can also be cloned into a single copy yeast vector such as pHR70 of Hans Ronne or pRS313, pRS314, pRS315 or pRS316 (Sikorski and Hieter, 1989). Alternatively, extra copies of SSOl or SS02 gene can also be integrated into the 15 yeast chromosomc, into the ribosomal RNA locus, for instance. For this purpose the ribosomal sequences of a suitable plasmid, e.g. plasmid pIRL9 (Hallborn et al., Pat. Appl.) are released, and cloned appropriately into BS+ vector, as shown in Fig. 7. The gene SSOl or SS02 coupled in between suitable yeast promoter and terminator regions, is released from the hybrid vector comprising the gene and cloned into the 20 plasmid obtained at the previous stage. From this resulting plasmid the expression cassette, flanked by ribosomal sequences can be released. This fragment is cotransformcd into a yeast with an autonomously replicating plasmid carrying a suitable marker for transformation. Hie plasmid can be later on removed from the cells containing the extra copies of SSOl or SS02 gene integrated in the chromosome 25 by cultivating the cells in non-selective conditions. This way, recombinant strains can be obtained which carry no extra foreign DNA such as bacterial vector sequences. If a polyploid yeast strain, such as VTT-A-63015, is used the gene can be integrated also to an essential locus such as the ADH1 or the PGK1 locus. 30 To express the SSO genes in Trichoderma the coding region of the Trichoderma sso gene is coupled for instance between the T. reesei cbhl promoter and terminator and the expression cassette is transformed into a Trichoderma strain producing for WO 94/08024 PCT/FI93/00402 instance mammalian antibodies or another foreign protein or into a strain producing EGIcore, another cellulase or a hydrolytic enzyme. Enhancement of secretion would be especially desired when the fungus is grown on glucose-containing media and for this purpose the sso gene(s) need to be expressed from constitutive promoters or 5 promoters functioning on glucose medium. For filamentous fungi the sso gene is preferably integrated into the genome using methods known in the art. Suitable promoters in addition to the cbhl promoter or promoter of the sso gene itself are for instance the other cellulase promoters, cbh2, 10 egll, egl2, or tefl, pgk, gpd, pki, the glucoamylase, a-amylase or the alcohol dehydrogenase promoter. In filamentous fungi transformation usually results in strains with varying copies of the sso gene integrated into the genome (Penttila et al., 1987) and from these the strain with optimal level of sso expression for growth and enhanced secretion can be screened. 15 An object of this invention is thus to provide SSO genes, especially the SSOl and SS02 genes of S. cerevisiae, as well as homologous gene(s) of Trichoderma reesei and other eukaryotic cells. The sequence of the genes can be determined from the plasmids carrying them by using e.g. the double stranded dideoxy nucleotide 20 sequencing method (Zagursky et al, 1986). The sequence of the SSOl gene of 5. cerevisiae is given as the SEQ ID NO. 1 and the sequence of the SS02 gene of S. cerevisiae is given as the SEQ ID NO. 3. Another object of this invention is to provide specific vectors comprising the SSO 25 genes. For yeast such a vector is either an autonomously replicating multicopy or a single copy plasmid or a vector capable of integrating into the chromosome, as described above. For Trichoderma such a vector is preferably a plasmid from which the expression cassette (promoter - gene - terminator) can be released by restriction enzymes to be integrated into the fungal genome. 30 Still another object of this invention is to provide yeast or other fungal strains as toll as eukaryotic cell lines containing extra copies of SSO genes either on replicating WO 94/08024 PCT/F193/00402 14 plasmid(s) or integrated into the chromosomes, which results in increased production of secreted proteins, such as yeast invertase or Trichoderma cellulases or other hydrolases. 5 Thus a method for constructing new eukaryotic cells capable of expressing enhanced levels of Sso protein(s) comprises: (a) isolating DNA sequence(s) coding for Sso protein(s) from a suitable donor organism; (b) constructing vectors) carrying at least one of the said DNA sequences; and 10 (c) transforming at least one of the vectors obtained to suitable host cells. Still another object of this invention is to provide eukaryotic cells which in addition to extra copies of SSO genes comprise a DNA sequence coding for a secreted foreign or endogenous protein, such as a-amylase, cellulase, or an antibody and are capable IS of expressing this protein. Thus a process for producing increased amounts of secreted foreign or endogenous protein(s) by overexpressing the SSO gene(s) is provided. This process comprises: (a) isolating DNA sequence(s) coding for the said protein(s) from a suitable 20 donor organism; (b) constructing a vector carrying at least one of the said DNA sequences; (c) transforming the vector obtained into a suitable host expressing enhanced levels of Sso protein(s) to obtain recombinant host cells; or alternatively, transforming the vector to a suitable host and re transforming this 25 transform ant with SSO or a gene homologous to SSO and screening for cells with enhanced production of the said protein(s); and (d) cultivating said recombinant host cells under conditions permitting expression of said protein(s). 30 A further object of this invention is to improve secretion by optimizing the Sso-protein level using different promoters and different copy numbers of the gene land combining the SSO genes with other genes involved in secretion, such as SEC1. WO 94/08024 PCT/FI93/00402 Thus the invention provides a process for producing increased amounts of secreted foreign or endogenous protcin(s), by overexpressing gene(s) interacting with the SSO gene, e.g. SEC1, in the presence of normal or increased amounts of the Sso protein(s), which process comprises: 5 (a) isolating DNA sequence(s) coding for the said protein(s) from suitable donor organism; (b) constructing a vector carrying at least one of the said DNA sequences; (c) transforming the vector obtained into a suitable host expressing normal or enhanced levels of Sso protein(s) and overexpressing other gene(s) 10 interacting with SSO gene, e.g. SEC1, to obtain recombinant host cells; or, alternatively, transforming the vector to a suitable host and re transforming this transformant with SSO or a gene homologous to SSO and by the gene interacting with SSO gene and screening for cells with enhanced production of the said protein(s); and 15 (d) cultivating said recombinant host cells under conditions permitting expression of said protein(s). Still another object of this invention is to provide a process for increased production of an endogenous secreted protein, the process comprising: 20 (a) transforming cells producing the said protein with a SSO gene or a gene homologous to SSO, alone or together with gene(s) interacting with the SSO gene, such as SEC1, (b) screening for transform ants producing enhanced level of the said protein thus obtaining recombinant cells for enhanced protein production, and 25 (c) cultivating said recombinant cells in conditions permitting expression of said protein. Still another object of this invention is to provide fungal strains which in addition to extra copies of SSO genes or their homologue comprise DNA sequence(s) coding for 30 hydrolytic enzyme(s) such as a-amylase and/or glucoamylase or lignocellulose hydrolyzing enzymes such as cellulase(s), hemicellulases or ligninases, which rcilder WO 94/08024 PCT/FI93/00402 16 the fungus capable of increased hydrolysis of, and/or enhanced growth on polymeric compounds such as starch or lignocellulose. Thus an efficient biomass production on said raw material or efficient hydrolysis of 5 said raw material is provided. This process comprises: (a) isolating DNA sequence(s) coding for endogenous or foreign hydrolytic enzyme(s) from a suitable donor organism; (b) constructing a fungal vector carrying at least one of the said DNA sequences; 10 (c) transforming the vector obtained into a suitable fungal host expressing expression of said hydrolytic enzyme(s). A process is also provided for efficient biomass production on a raw material or efficient hydrolysis of a raw material, by overexpressing genes interacting with the 20 SSO gene, e.g. SEC1, in the presence of normal or increased amounts of the Sso protein(s). This process comprises: enhanced levels of Sso protein(s) to obtain recombinant host cells; or alternatively, transforming the vector to a suitable host and re transforming this transformant with SSO or a gene homologous to SSO and screening for cells with enhanced production of the said enzyme(s); and 15 (d) cultivating said recombinant host cells under conditions permitting (a) isolating the DNA sequence(s) coding for endogenous or foreign hydrolytic enzyme(s) from a suitable donor organism; 25 (b) constructing a vector carrying at least one of the said DNA sequences; 30 (c) transforming the vector obtained to a suitable host expressing enhanced levels of proteins interacting with the Sso protein(s) in the presence of normal or increased amounts of the Sso protein(s) to obtain recombinant host cells, or, alternatively, transforming the vector to a suitable host and ^transforming this transformant with SSO gene or a gene homologous to SSO and with the gene(s) WO 94/08024 PCT/FI93/00402 17 interacting with SSO gene, such as SEC1, and screening for cells with enhanced production of the said enzyme(s); and (d) cultivating said recombinant host cells under conditions permitting expression of said hydrolytic enzyme(s). 5 Possible applications of said recombinant cells are e.g. in single cell production, improved alcohol production or in processes where efficient hydrolysis of raw material is desired. 10 EXPERIMENTAL Example 1: Cloning of the coding region of SSOl and SS02 genes from Saccharomyces cerevisiae. 15 The SSOl and SS02 genes were isolated as suppressors of the temperature-sensitive defect of seel -1 mutant (Novick and Scheckman, 1979; Novick etal., 1980). The S. cerevisiae strain sf750-14Da (a secl-1 his4 ura3-52 trpl~289 leu2-3 le.u2-112) (obtained from Randy Scheckman, University of California, Berkeley, CA) was transformed (Ito et al, 1983) by yeast cDNA library constructed by McKnight and 20 McConaughy (1983) from strain X2180-1B on a 2\i based plasmid, pMACSSl, containing TRP1 as a selection marker, and selected for Trp-prototrophy at 37°C. As the growth of the transfonnants was refractory at 37°C, further work was done at 36.5 or 3S°C temperatures which still are non-permissive for secl-1. DNA isolated (Keranen, 1986) from four yeast transfonnants which showed co-segregation of the 25 Trp+ phenotype and growth at 36.5°C was transferred into E. coli (Hanahan, 1983). Plasmid DNA isolated from E. coli transfonnants was used to re-transform the seel -1 strain of S. cerevisiae. Efficient transformation for growth at 36.5°C was obtained. Restriction enzyme 30 analysis of the plasmids indicated that two different sequences were recovered from the cDNA library used. The insert DNA from the two different clones, 1 and 7, was sequenced using the double stranded dideoxy method (Zagursky et al, 1986) and Otfica Fw i ii'i*•*> »"*« Applies .ion PCT/ FI 93/00402 21 -12- 1994 18 suitable subclones constructed with standard recombinant DNA methods (Maniatis et al., 1982) or specific primers. The two clones contained an open reading frame of 870 nucleotides (clone 1) and 885 nucleotides (clone 7), respectively. As the deduced amino acid sequences did not represent that of the Seel protein (Aalto et al, 1991) the new genes were named SSOl and SS02 (Suppressor of Seel fine). The SSOl and SS02 coding sequences and the deduced amino acid sequences are given in SEQ ID NO: 1 and SEQ ID NO: 3, respectively. The plasmids carrying the SSOl and SS02 genes were named YEpSSOl and YEpSS02, respectively and are shown in Figs. 1A and IB. Example 2: Overexpression of the Sso2 protein in yeast transformed with YEpSS02. The yeast strain sf750-14D transformed with the control plasmid pMA56 (A) (Ammerer, 1983) or with YEpSS02 (B) were grown in synthetic complete medium (Sherman et al. 1983) lacking Tip. Yeast cell lysates were prepared in the presence of SDS as described by Keranen (1986). Ten |xg of total yeast protein present in the lysates were separated by SDS-PAGE and analyzed by Western blotting using polyclonal antibodies made in rabbit against the Sso2 protein and alkaline phosphatase conjugated goat anti-rabbit IgG for detection. As shown in Fig. 2, greatly increased amount of Sso2 protein was seen in the YEpSS02 transformant. Example 3: Enhanced production of secreted heterologous protein, Bacillus a-amylase in yeast strain s(750-14D overexpressing either SSOl or SS02. The yeast strain sf750-14Da harboring either SSOl or SS02 gene on the multicopy plasmids YEpSSOl or YEpSS02, respectively, were transformed with a multicopy plasmid YEpaaS containing Bacillus a-amylase gene ligated between the ADH1 promoter and terminator (Ruohonen et al, 1987), modified for more efficient expression by deleting predicted inhibitory sequences 5* to the promoter element (Ruohonen et al, 1991; Ruohonen et al, manuscript in preparation, a). The yeast strains obtained containing YEpSSOl and YEpaaS (VTT-C-92072) or YEpSS02 SHEET WO 94/08024 PCT/FI93/00402 ► 19 and YEpaa5 (VTT-C-92073) were grown in scicctivc medium at 24°C and secretion of a-amylase into the culture medium was monitored by measuring the a-amylase activity using the Phadebas amylase test (Pharmacia Diagnostics AB, Sweden). These strains VTT-C-92072 and VTT-C-92073 were deposited at the Deutsche Sammlung S von Mikroorganismen und Zcllkulturcn GmbH (DSM) on 30 September 1992 with the accession numbers DSM 7253 and 7254, respectively. As shown in Fig. 3, increased a-amylase activity was obtained in strains which carried either SSOl (*) or SS02 (■) on the multicopy plasmid compared with the untransformed control strain (•). Segregation of YEpSSOl (A) or YEpSS02 (□) off from the transfonnants 10 reduced the a-amylase secretion to the control level proving that the increased secretion is due to the presence of the SSO gene containing plasmids in the transfonnants. Increased amount of a-amylase protein in the culture medium was detected by Western blotting (Fig. 4). Symbols as for Fig. 3., S = standard (Bacillus a-amylase). 15 Example 4: Enhanced production of secreted foreign protein, Bacillus a-amylase and an endogenous protein, invertase in yeast strain DBY746 overexpressing SS02. 20 The S. cerevisiae strain DBY746 (a kis3Al leu2-3 leu2-112 ura3-52 trpl-289 cgh*) (obtained from David Botstein, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA) harboring the plasmid YEpaa6 containing Bacillus a-amylase gene iigated between the ADH1 promoter and terminator (Ruohonen et al, 1987), modified for more efficient expression by deleting predicted inhibitory 25 sequences 5' to the promoter element (Ruohonen et al, 1991; Ruohonen et al, manuscript in preparation, a) was transformed either with YEpSS02 or with the control plasmid pMA56 (Ammeter, 1983). The transform ants were grown in selective medium at 30°C and secretion of a-amylase into the culture medium was monitored by measuring the a-amylase activity using the Phadebas amylase test (Pharmacia 30 Diagnostics AB, Sweden). As shown in Fig. 5, increased a-amylase activity was obtained in the strain which carried SS02 (A) on the multicopy plasmid' compared with the control strain transformed with the control plasmid without SSO gene (O). WO 94/08024 PCT/FI93/00402 > 20 No difference was observed in the yeast growth between the control transformant (•) and SS02 transformant (*). Overexpression of SSOl increased the secretion of a-amyiase in a similar manner. Secretion of the endogenous protein, invertase, was also enhanced under these conditions measured at late logarithmic to early stationary 5 growth phase. The secreted invertase activity in the YEpSS02 transformant was 1.4 times that in the control transformant containing pMA56. As the enhancing effect of SSO overexpression on a-amylase secretion is more pronounced later during the growth, also the invertase secretion should be more enhanced at later time points. 10 Removal of the predicted inhibitory sequences on the ADH1 promoter (see above) used for expression of the SS02 in YEpSS02 resulted in prolonged expression of SS02 and prolonged existence of increased level of the Sso2 protein and consequently even higher final levels of the Bacillus a-amylase secreted into the medium. Expression of SS02 on a single copy plasmid from this modified ADH1 15 promoter also resulted in increased levels of the Sso2 protein and enhanced secretion of a-amylase. Example 5: Enhanced production of secreted foreign protein, Bacillus a-amylase in yeast overexpressing SEC1 in combination with normal 20 or increased levels of functional Sso proteins. Hie S. cerevisiae strain DBY746 harboring the plasmid YEpaa6 containing Bacillus a-amylase gene ligated between the ADH1 promoter and terminator (Ruohonen et al, 1987), modified for more efficient expression by deleting predicted inhibitory 25 sequences 5' to the promoter element (Ruohonen et al, 1991; Ruohonen et al, manuscript in preparation, a) was transformed either with a multicopy plasmid YEpSECl expressing the SEC1 gene or with the control plasmid YEp24H (Aalto et al., 1991; Ruohonen et al> manuscript in preparation, b). The transfonnants were grown in selective medium at 30°C and secretion of a-amylase into the culture 30 medium was monitored by measuring the a-amylase activity using the Phadebas amylase test (Pharmacia Diagnostics AB, Sweden). As shown in Fig. 6, increased*a-amylase activity was obtained in the strains which carried SEC1 on a multicopy WO 94/08024 PCT/FI93/00402 > 21 plasmid (□) compared with the strains transformed with the vector without SEC1 gene (O). No difference was observed in the growth between the transfonnants. Overexpression of both Seclp and Sso2p at the same time enhanced a-amylase 5 secretion even further. The plasmids expressing the SSO genes are available at VTT, Biotechnical Laboratory, Espoo, Finland. Example 6: Isolation of the Trichoderma sso genes by expression in yeast and their expression in Trichoderma 10 A yeast expression gene bank prepared from the T. reesei strain QM9414 as described (Buchert et al., FI Pat Appl. 922373) was transformed into the Saccharomyces cerevisiae strain H458 (Aalto et al., 1993) (a SUC2 ade2-l canl -100 his3-11,15 leu2-3,112 trpl-1 ura 3-1 ssol-6l::URA3 sso2"62::ku2:: ■ «-. 15 (GALl:ssol,HIS3)) by selecting for Ura-prototrophy on a galactose medium The transfonnants were transfened onto glucose medium and the plasmid was rescued from the growing colonies and re transformed into the above mentioned strain to verify the complementation. A clone was obtained showing capability to rescue depletion of the Sso proteins on glucose medium and the conesponding plasmid was 20 named pMS51. The S. cerevisiae strain obtained, carrying ihe plasmid pMS51 was deposited at the Deutsche Sammlung von Mikvoorganismen und Zellkulturen GmbH (DSM) on 5 October 1993 with the accession number DSM 8604. The chromosomal copy of the gene is isolated from a genomic cosmid library (Mantyla et al., 1992) by using the 5' end of the cDNA clones as a probe, prepared by PCR. The cosmid is 25 isolated from the clones giving a signal, and those corresponding to the above mentioned cDNA are transformed into a T. reesei (Penttila et al, 1987) strain producing CBHI-Fab molecules VTT-D-91418 (CBS 287.91) described in Nyyss6nen et al., (Pat. Appl.). Production of CBHI-Fab is studied from the extracellular medium on Solca-floc medium (according to Nyyss6nen et al., Pat. 30 Appl.). 94/08024 PCT/FI93/00402 22 Example 7: Isolation of fungal sso genes by heterologous hybridization Genomic DNA from the fungal species Saccharirxyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Pichia stipitis, Aspergillus 5 nidulans and Trichoderma reesei were isolated, digested with the HindYll restriction enzyme, separated electrophoretically in an 0.8% agarose gel and blotted on a nylon filter. Southern hybridization of the filter was carried out at different stringencies using the yeast SSOl gene coding region as a probe. Hybridization in a mixture containing 30 % formamide, 6xSSC, 10 x Denhardt's, 0.5% SDS, 100 fig/ml herring 10 sperm DNA and 10 iigfrnl polyA at 35 °C and washing 2 x 30 minutes in 2xSSC, 0.1% SDS at 42 °C revealed several hybridizing bands in DNA derived from S. cerevisiae, K. lactis, P. stipitis and 71 reesei (Fig. 8). When hybridization was performed in less stringent conditions, hybridization was obscrvrd also with S. pombe DNA. A genomic T. reesei gene library constructed in the XEMBL3 (Frischauf et al., 15 1983) vector was hybridized by the procedure described above. Clones giving hybridization signals were purified and their hybridizing regions were mapped by digestions and Southern hybridizations of their DNA. The three hybridizing k clones were designated TSSOa, TSSOb and TSSOc. These clones were deposited at the Deutsche Sammlvng von Mikroorganismen und Zellkulturen GmbH (DSM) on 5 20 October 1993 with the accession numbers DSM 8601, DSM 8602 and DSM 8603, respectively. WO 94/08024 PCI/FI93/00402 ) 23 Deposited microorganisms The following microorganisms were deposited according to the Budapest Treaty at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), 5 Mascheroder Weg lb, D-3300 Braunschweig, Germany. Strain Deposition number Deposition date Saccharomyces cerevisiae DSM 7253 30 September 1992 10 VTT-C-92072 carrying the plasmid YEpSSOl Saccharomyces cerevisiae DSM 7254 30 September 1992 VTT-C-92073 carrying the 15 plasmid YEpSS02 Saccharomyces cerevisiae H458 (VTT-C-93002) carrying the plasmid pMSSl DSM 8604 5 October 1993 20 Bacteriophage X strain TSSOa (VTT-H-93001) DSM 8601 5 October 1993 Bacteriophage X strain 25 TSSOb (VTT-H-93002) DSM 8602 5 October 1993 Bacteriophage "K strain TSSOc (VTT-H-93003) DSM 8603 5 October 1993 WO 94/08024 PCT/F193/00402 24 References 5 Aalto, M.K., Keranen, S. and Ronne, H. 1992. A family of proteins involved in intracellular transport. Cell 68, 181-182. 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Vanoni, M., Porro, D., Martegani, E. and Alberghina, L. 1989. Secretion of Escherichia coli j3-galactosidase in Saccharomyces cerevisiae using the signal Aft/iEM5cD SHEET I I ' O W P^7 i;',. PC!/ Fl 9 3 / 0 0 4 21 -12- 1994 28 sequence from the glucoamylase-encoding STA2 gene. Biochem. Biophys. Res. Commun. 164, 1331-1338. Ward, M., Wilson, L.J., Kodama, K.H., Rcy, M.W. and Berka, R.M. 1990. Improved production of calf chymosin in Aspergillus by expression as a giucoamylase-chymosin fusion. Bio/Technology 8, 435-440. Wilson, D.W., Wilcox, CA., Flynn, G.C., Chen, E., Unang, W-J., Henzel, WJ., Block, M.R., Ullrich, A. and Rothman, J.E., 1989. A fusion protein required for vehicle-mediated transport in both mammalian cells and yeasts. Nature 339, 355-359. f, Jfcagursky, R.J., Berman, M.L., Baumeister, K. and Lomax, N. 1986. Rapid and easy sequencing of large linear double stranded DNA and supercoiled plasmid DNA. Gene Anal. Techn. 2, 89-94. Zaraoui, A., Touchot, N., Chardin, P. and Tavitian, A. 1989. The human Rab genes encode family or GTP-binding proteins related to yeast YTP1 and SEC4 products involved in secretion. J. Biol. Chem. 264, 12394-12401. AMENDED SHEET 256 425 29 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule 13bu) A. Tbe indications made below relate to tbe microorganism referred lo in tbe description 18 .line 32 on page B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet El Name of depositary institution Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) Date of deposit 30 September 1992 Accession Number DSM 7253 C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet |X | Address of depositary institution (includingpostal code and country) Mascheroder Wcg lb, D-3300 Braunschweig, Germany In respect of those designations in which a European patent or a patent in Finland or Norway is sought, a sample of the deposited microorganism will be made available until the publication of the mention of the grant of the European patent or the corresponding information concerning the patent in Finland or Norway or until the date on which the application has been refused or withdrawn or is deemed to be withdrawn, only by the issue of such a sample lo an expert nominated by the person requesting the sample (Rule 28(4) EPC and the corresponding regulations in Finland and Norway). D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (ifthe indications arc not for ell designated Stales) N.Z. KM i'NT OEfKJfc 2 3 MAY 1996 B6C»vet) E. SEPARATE FURNISHING OF INDICATIONS (leave blank ifnot applicable) Tbe indications listed below will be submittal to tbe Inienwtional Bureau later (specify the general nature oftheiniications e.j, 'Accession Number of Deposit") For receiving Office use only Q This sbeet was received with tbe international application Authorized officer /Ym TVluhoiC- For International Bureau use only □ This sbeet was received by tbe International Bureau on: Authorized officer Form PCT/RO/134 (July 1992) 30 256 425 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule 13 bis) A. Tbe indications nude below relate to tbe microorganism referred to in the description 19 .line _J on page B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sbeet El Name of depositary institution Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) Address of depositary institution (includingpostal code and country) Mascheroder Weg lb, D-3300 Braunschweig, Germany Date of deposit 30 September 1992 Accession Number DSM 7254 C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sbeet | XI In respect of those designations in which a European patent or a patent in Finland or Norway is sought, a sample of the deposited microorganism will be made available until the publication of the mention of the grant of the European patent or the corresponding information concerning the patent in Finland or Norway or until the date on which the application has been refused or withdrawn or is deemed to be withdrawn, only by the issue of such a sample to an expert nominated by the person requesting the sample (Rule 28(4) EPC and the corresponding regulations in Finland and Norway). D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for at! designated States) HZ. PATENT OTFKJfc 1 2 3 MAY 1996 wccirnvfrfl E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable} Tbe indications listed below will be submitted to tbe International Bureau later (specify Ae general nature oftheindicaiions e.g^ 'Accession Number of Deposit") For receiving Officc use only \K\ Tb" sheet was received with tbe international application Authorized officer Form PCI7R0/134 (July 1992) For International Bureau use only □ This sbeet was received by tbe International Bureau on: Authorized officer 31 256425 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule 13bis) A. Tbe indications made below relate to tbe microorganism referred to in tbe description on page . £2 t |jnc 1j8 B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sbeet |X | Name of depositary institution Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) Address of depositary institution (includingpostal code and country) Maschcroder Weg lb, D-3300 Braunschweig, Germany Date of deposit Accession Number 5 October 1993 DSM 8601 C. ADDITIONAL INDICATIONS (tern ve blank if net applicable) This information is continued on an additional sbeet (x I In respect of those designations in which a European patent or a patent in Finland or Norway is sought, a sample of the deposited microorganism will be made available until the publication of the mention of the grant of tbe European patent or the corresponding information concerning the patent in Finland or Norway or until tbe date on which tbe application has been refused or withdrawn or is deemed to be withdrawn, only by the issue of such a sample to an expert nominated by the person requesting the sample (Rule 28(4) EPC and the corresponding regulations in Finland and Norway). D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (//the indications are net for ell designated Stales) M.Z. PATENT E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not epplicabl 2 3 Wv 199B REC-* Tbe indications listed below will be submitted to the International Bureau later (specify the general nature vf the indications e.g^ 'Accession Number of Deposit") For receiving Office use only IX 1 This sbeet was received with tbe international application Authorized officer fyiufiTQlbl&ft- For International Bureau use only □ This sbeet was received by tbe International Bureau on: Authorized officer Form PCI7RO/134 (July 1992) 32 25 6 425 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule \ii is) A. Tbe indications made below relate to tbe microorganism referred to in tbe description on page ^ .. . line ^ ^ B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sbeet jx~| Name of depositary institution Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) Address of depositary institution (including postal code a.nd country) Maschereder Weg lb, D-3300 Braunschweig, Germany Date of deposit 5 Octobcr 1993 Accession Number DSM 8602 C. ADDITIONAL INDICATIONS (leave blank if net applicable) This information is continued on an additional sbeet |X 1 In respect of those designations in which a European patent or a patent in Finland or Norway is sought, a sample of tbe deposited microorganism will be made available until the publication of the mention of the grant of the European patent or the corresponding information concerning the patent in Finland or Norway or until the date on which the application has been refused or withdrawn or is deemed to be withdrawn, only by the issue of such a sample to an expert nominated by tbe person requesting the sample (Rule 28(4) EPC and the corresponding regulations in Finland and Norway). D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (ifthe indications irt not for ell designated States) N.Z. p KTr.nr OPRCE 2 3 MAY 1996 RfiCffiVBD E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) Tbe indications listed below will be submitted to tbe International Bureau later (specify the general nature ofihe indications 'Accession Number of Deposit") For receiving Office use only bCl Tbis sbeet was received with tbe international application Authorized officcr /}f/#0 TQLufooct— For International Bureau use only □ Tbis sbeet was received by tbe International Bureau on: Authorized officer Form PCT/RO/134 (July 1992) 33 256423 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule 13 bis) A. Tbe indications made below relate to tbe microorganism referred to in tbe description 22 ,.18 on page , line B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sbeet 0 Name of depositary institution Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) Address of depositary institution (includingpostal codc and country) Mascheroder Wcg lb, D-3300 Braunschweig, Germany Date of deposit 5 October 1993 Accession Number DSM 8603 C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet |X | In respect of those designations in which a European patent or a patent in Finland or Norway is sought, a sample of the deposited microorganism will be made available until the publication of the mention of the grant of tbe European patent or the corresponding information concerning the patent in Finland cr Norway or until the date on which tbe application has been refused or withdrawn or is deemed to be withdrawn, only by the issue of such a sample to an expert nominated by the person requesting the sample (Rule 28(4) EPC and the corresponding regulations in Finland and Norway). D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for til designated Stoics) N.Z. PATENT O^FIOE 2 3 MAY 1996 HECSIVK) SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) The indications listed below will be submitted to tbe International Bureau later (specify the general nature ofihcuuBcetiom eg, 'Accession Number of Deposit") For receiving Office use only |Vf This sbeet was received with tbe international application Autborized officer /Wo Form PCT/RO/134 (July 1992) For International Bureau use only | j Tbis sheet was received by tbe International Bureau on: Autborized offiet. 34 25 6 425 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule I3bis) A. Tbe indications made below relate lo tbe microorganism referred to in tbe description 21 t line 20 on page B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sbeet □ Name of depositary institution Dcutschc Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) Address of depositary institution (including postal code end country) Mascheroder Weg lb, D-3300 Braunschweig, Germany Date of deposit 5 October 1993 Accession Number DSM 8604 C. ADDITIONAL INDICATIONS (leave blank if not applicable) Tbis information is continued on an additional sbeet |X| In respect of those designations in which a European patent or a patent in Finland or Norway is sought, a sample of the deposited microorganism will be made available until the publication of the mention of the grant of tbe European patent or the corresponding information concerning the patent in Finland or Norway or until the date on which the application has been refused or withdrawn or is deemed to be withdrawn, only by tbe issue of such a sample to an expert nominated by tbe person requesting the sample (Rule 28(4) EPC and the corresponding regulations in Finland and Norway). D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for til designated States) N.Z. PATENT OFFKX 2 3 MAY 1996 HLUJWbU E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not appKMtof Tbe indications listed below will be submitted to tbe international Bureau \z\a (specify the general nature of the indications c.g^ "Accession Number of Deposit") For receiving Office use only j Tl>is sbeet was received with tbe international application Authorized ofDcer /waTUuiotc* For International Bureau use only □ Tbis sheet was received by tbe International Bureau on: Authorized officer Form PCT/RO/134 (July 1992) C. J 6 4 2 5 35 SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Valtion teknillinen tutkimuskeskus (B) STREET: Vuorimiehentie 5 (C) CITY: Espoo (E) COUNTRY: Finland (F) POSTAL CODE (ZIP): FIN-02150 (ii) TITLE OF INVENTION: Increased production of Becreted proteins by recombinant eukaryotic eel1b (iii) NUMBER OF SEQUENCES: 4 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy diBk (B) COMPUTERS IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version /1.25 (EPO) (vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: FI 92 4494 (B) FILING DATE: 06-OCT-1992 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 870 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Saccharomyces cerevisiae (B) STRAIN: X2180-1B (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..870 N.Z. PAT£KT OmOE 2 3 MAY 1936 RSC©V«E8 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: ATG AGT TAT AAT AAT CCG TAC CAG TTG GAA ACC CCT TTT GAA GAG TCA Met Ser Tyr Asn Asn Pro Tyr Gin Leu Glu Thr Pro Phe Glu Glu Ser 1 5 10 15 48 TAC GAG TTG GAC GAA GGT TCG AGC GOT ATC GGT GCT GAA GGC CAC GAT Tyr Glu Leu Asp Glu Gly Ser Ser Ala lie Gly Ala Glu Gly His Asp 20 2S 30 96 TTC GTG GGC TTC ATG AAT AAG ATC AGT CAA ATC AAT CGC GAT CTC GAT Phe Val Gly Phe Met Asn Lys lie Ser Gin lie Asn Arg Asp Leu Asp 35 40 45 144 AAG TAC GAC CAT ACC ATC AAC CAG GTC GAT TCT TTG CAT AAG AGG CTA Lys Tyr Asp His Thr lie Asn Gin Val Asp Ser Leu His Lys Arg Leu 50 55 60 192 C j 0 'i 2 5 36 CTG ACC GAA GTT AAT GAG GAG CAA GCA AGT CAC TTA AGG CAC TCC CTG Leu Thr Glu Val Asn Glu Glu Gin Ala Ser His Leu Arg His Ser Leu 65 70 75 80 240 GAC AAC TTC GTC GCA CAA GCC ACG GAC TTG CAG TTC AAA CTG AAA AAT Asp Aen Phe Val Ala Gin Ala Thr Asp Leu Gin Phe Lys Leu Lys Asn 85 90 95 288 GAG ATT AAA AGT GCC CAA AGG GAT GGG ATA CAT GAC ACC AAC AAG CAA Glu lie Lys Ser Ala Gin Arg Asp Gly lie His Asp Thr Asn Lys Gin 100 105 110 336 GCT CAG GCG GAA AAC TCC AGA CAA AGA TTT TTG AAG CTT ATC CAG GAC Ala Gin Ala Glu Asn Ser Arg Gin Arg Phe Leu Lys Leu lie Gin Asp 115 120 125 384 TAC AGA ATT GTG GAT TCC AAC TAC AAG GAG GAG AAT AAA GAG CAA GCC Tyr Arg lie Val Asp Ser Asn Tyr Lys Glu Glu Asn Lys Glu Gin Ala 130 135 140 432 AAG AGG CAG TAT ATG ATC ATT CAA CCA GAG GCC ACC GAA GAT GAA GTT Lys Arg Gin Tyr Met lie lie Gin Pro Glu Ala Thr Glu Asp Glu Val 145 150 155 160 480 GAA GCA GCC ATA AGC GAT GTA GGG GGC CAG CAG ATC TTC TCA CAA GCA Glu Ala Ala lie Ser Asp Val Gly Gly Gin Gin lie Phe Ser Gin Ala 165 170 175 528 TTG TTG AAT GCT AAC AGA CGT GGG GAA GCC AAG ACT GCT CTT GCG GAA Leu Leu Asn Ala Asn Arg Arg Gly Glu Ala Lys Thr Ala Leu Ala Glu 180 185 190 576 GTC CAG GCA AGG CAC CAA GAG TTA TTG AAA CTA GAA AAA TCC ATG GCA Val Gin Ala Arg His Gin Glu Leu Leu Lys Leu Glu Lys Ser Het Ala 195 200 205 624 GAA CTT ACT CAA TTG TTT AAT GAC ATG GAA GAA CTG GTA ATA GAA CAA Glu Leu Thr Gin Leu Phe Asn Asp Met Glu Glu Leu Val lie Glu Gin 210 215 220 672 CAA GAA AAC GTA GAC GTC ATC GAC AAG AAC GTT GAA GAC GCT CAA CTC Gin Glu Asn Val Asp Val lie Asp Lys Asn Val Glu Asp Ala Gin Leu 225 230 235 240 720 GAC GTA GAA CAG GGT GTC GGT CAT ACC GAT AAA GCC GTC AAG AGT GCC Asp Val Glu Gin Gly Val Gly His Thr Asp Lys Ala Val Lys Ser Ala 245 250 255 768 AGA AAA GCA AGA AAG AAC AAG ATT AGA TGT TGG TTG ATT GTA TTC GCC Arg Lys Ala Arg Lys Asn Lys lie Arg Cys Trp Leu lie Val Phe Ala 260 265 270 816 ATC ATT GTA GTC GTT GTT GTT GTC GTT GTT GTC CCA GCC GTT GTC AAA lie lie Val Val Val Val Val Val Val Val Val Pro Ala Val Val Lys 275 280 285 864 ACG CGT Thr Arg 290 870 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 290 amino acids N.Z. PATENT OFFICE 2 3 MAY 1996 HSCBVED 2 5 6 4 2 5 37 (B) TYPE: amino acid (D) TOPOLOGYt linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Ser Tyr Asn Asn Pro Tyr Gin Leu Glu Thr Pro Phe Glu Glu Ser 15 10 15 Tyr Glu Leu Asp Glu Gly Ser Ser Ala lie Gly Ala Glu Gly His Asp 20 25 30 Phe Val Gly Phe Met Asn Lys lie Ser Gin lie Asn Arg Asp Leu Asp 35 40 45 Lys Tyr Asp His Thr Ila Asn Gin Val Asp Ser Leu His Lys Arg Leu 50 55 60 Leu Thr Glu Val Asn Glu Glu Gin Ala Ser His Leu Arg His Ser Leu 65 70 75 80 Asp Asn Phe Val Ala Gin Ala Thr Asp Leu Gin Phe Lys Leu Lys Asn 85 90 95 Glu lie Lys Ser Ala Gin Arg Aap Gly lie His Asp Thr Asn Lys Gin 100 105 110 Ala Gin Ala Glu Asn Ser Arg Gin Arg Phe Leu Lys Leu lie Gin Asp 115 120 125 Tyr Arg lie Val Asp Ser Asn Tyr Lys Glu Glu Asn Lys Glu Gin Ala 130 13S 140 Lys Arg Gin Tyr Met lie lie Gin Pro Glu Ala Thr Glu Asp Glu Val 145 150 155 160 Glu Ala Ala lie Ser Asp Val Gly Gly Gin Gin lie Phe Ser Gin Ala 165 170 175 Leu Leu Asn Ala Asn Arg Arg Gly Glu Ala Lys Thr Ala Leu Ala Glu 180 185 190 Val Gin Ala Arg His Gin Glu Leu Leu Lys Leu Glu Lys Ser Met Ala 195 200 205 Glu Leu Thr Gin Leu Phe Asn Asp Met Glu Glu Leu Val lie Glu Gin 210 215 220 Gin Glu Asn Val Asp Val lie Asp Lys Asn Val Glu Asp Ala Gin Leu 225 230 235 240 Asp Val Glu Gin Gly Val Gly His Thr Asp Lys Ala Val Lys Ser Ala 245 250 255 Arg Lys Ala Arg Lys Asn Lys lie Arg Cys Trp Leu lie Val Phe Ala 260 265 270 lie lie Val Val Val Val Val Val Val Val Val Pro Ala Val Val Lys 275 280 285 Thr Arg 290 38 256 425 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 885 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Saccharomyces cerevisiae (B) STRAIN: X2180-1B (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..885 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: ATG AGC AAC GCT AAT CCT TAT GAG AAT AAC AAT CCG TAC GCT GAA AAC 48 Met Ser Asn Ala Asn Pro Tyr Glu Asn Asn Asn Pro Tyr Ala Glu Asn 1 5 10 15 TAT Tyr GAA ATG CAA Glu Met Gin 20 GAG Glu GAC TTG Asp Leu AAC Asn AAT Asn 25 GCT Ala CCT Pro ACT Thr GGT Gly CAC His 30 TCA Ser GAT Asp 96 GGT Gly AGC GAC GAT Ser ABp ABp 35 TTC Phe GTA GCT Val Ala TTT Phe 40 ATG Met AAC Asn AAG Lys ATC lie AAC ABn 45 TCA Ser ATA lie AAT ABn 144 GCT Ala AAC TTG TCC Asn Leu Ser 50 AGG Arg TAC GAA AAC Tyr Glu Asn 55 ATT lie ATC lie AAC ABn CAA Gin 60 ATT lie GAT Asp GCG Ala CAA Gin 192 CAC His 65 AAA GAC CTA Lys Asp Leu CTT Leu ACT CAA Thr Gin 70 GTG Val AGT Ser GAG Glu GAA Glu 75 CAG Gin GAG Glu ATG Met GAA Glu TTG Leu 80 240 AGA Arg CGT TCT TTG Arg Ser Leu GAC Asp 85 GAT TAC Asp Tyr ATC He TCT Ser CAG Gin 90 GCC Ala ACA Thr GAT Asp TTG Leu CAG Gin 95 TAT Tyr 288 CAA Gin TTG AAA GCG Leu Lys Ala 100 GAT Asp ATC AAA lie Lys GAT ASP GCC Ala 105 CAG Gin AGA Arg GAC Asp GGA Gly TTG Leu 110 CAC His GAC Asp 336 TCT Ser AAT AAA CAG Asn Lys Gin 115 GCA Ala CAA GCT Gin Ala GAA Glu 120 AAT Asn TGC Cys AGA Arg CAG Gin AAA Lys 125 TTC Phe TTA Leu AAA Lys 384 TTA Leu ATT CAA GAC lie Gin Asp 130 TAC Tyr AGA ATT Arg lie 135 ATC lie GAT Asp TCT Ser AAC ABn TAC Tyr 140 AAA Lys GAA Glu GAA Glu AGC Ser 432 AAA Lys 145 GAG CAG GCG Glu Gin Ala AAG Lys AGA CAG TAC Arg Gin Tyr 150 ACA Thr ATT lie ATC lie 155 CAA Gin CCG Pro GAA Glu GCC Ala ACT Thr 160 480 GAC Asp GAA GAA GTG Glu Glu Val GAA Glu 165 GCC GCC Ala Ala ATC lie AAC Asn GAT Asp 170 GTC Val AAT Asn GGC Gly CAG Gin CAG Gin 175 ATC lie 528 N.Z * 2 3 MAY 1996 RECEIVED 256425 39 TTT Phe TCC ser CAA Gin GCG Ala 180 TTG Leu CTA Leu AAC Asn GCC Ala AAT Asn 185 AGA Arg CGT GGT GAG Arg Gly Glu GCC Ala 190 AAG Lys ACA Thr 576 GCA Ala TTC Leu GCC Ala 195 GAA Glu GTA Val CAG Gin GCT Ala AGA Arg 200 CAT Hia CAA Gin GAG TTG Glu Leu TTG Leu 205 AAG Lys TTG Leu GAA Glu 624 AAA Lys ACA Thr 210 ATG Met GCT Ala GAA Glu CTT Leu ACC Thr 215 CAA Gin TTG Leu TTC Phe AAT GAC ATG Asn Asp Met 220 AAA Lys GAG Glu TTG Leu 672 GTC Val 225 ATC lie GAA Glu CAA Gin CAA Gin GAA Glu 230 AAT Asn GTG Val GAT Asp GTC Val ATT GAC AAA lie Asp Lys 235 AAC Asn GTC Val GAA Glu 240 720 GAC ABp GCT Ala CAG Gin CAA GAT GTA Gin Asp Val 245 GAG Glu CAA Gin GGT Gly GTG Val 250 GGT CAC Gly His ACC Thr AAC Asn AAG Lys 255 GCC Ala 768 GTT Val AAG Lys AGT Ser GCC AGA AAA Ala Arg Lys 260 GCA Ala AGA Arg AAA Lys 265 AAC Asn AAA ATA LyB lie AGA Arg TGT Cys 270 TTG Leu ATC lie 816 ATC lie TGC Cys TTT Phe 275 ATT lie ATC lie TTT Phe GCT Ala ATT lie 280 GTT Val GTT Val (JTl: GTT Val Val GTG Val 285 GTT Val GTT Val CCA Pro 864 TCC GTT GTG GAA ACA ACA AAG 885 Ser Val Val Glu Thr Arg Lys 290 295 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 295 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Met Ser Asn Ala Asn Pro Tyr Glu Aan Asn Asn Pro Tyr Ala Glu Asn IS 10 15 Tyr Glu Met Gin Glu Asp Leu Asn Aen Ala Pro Thr Gly His Ser ABp 20 25 30 Gly Ser Asp Asp Phe Val Ala Phe Met Asn Lys lie Asn Ser lie Asn 35 40 45 Ala Aon Leu Ser Arg Tyr Glu Asn lie lie Asn Gin lie Asp Ala Gin 50 55 60 His Lys Asp Leu Leu Thr Gin Val Ser Glu Glu Gin Glu Met Glu Leu 65 70 75 80 Arg Arg Ser Leu Asp Asp Tyr lie Ser Gin Ala Thr Asp Leu Gin Tyr 85 90 95 Gin Leu Lys Ala Asp lie Lye Asp Ala Gin Arg Asp Gly Leu His Asp 100 10* , llfl N.Z >* 'TENT OmQE 2 3 MAY 1996 RECEIVED 40 2 5 6 4 2 5 Ser Asn Lye Gin Ala Gin Ala Glu Aen Cys Arg Gin Lys Phe Leu Lys 115 120 125 Leu lie Gin Asp Tyr Arg lie lie Asp Ser Asn Tyr Lys Glu Glu Ser 130 135 140 Lys Glu Gin Ala Lys Arg Gin Tyr Thr lie lie Gin Pro Glu Ala Thr 145 150 155 160 Asp Glu Glu Val Glu Ala Ala lie Asn ABp Val Asn Gly Gin Gin lie 165 170 175 Phe Ser Gin Ala Leu Leu Asn Ala Asn Arg Arg Gly Glu Ala Lys Thr 180 185 190 Ala Leu Ala Glu Val Gin Ala Arg His Gin Glu Leu Leu Lys Leu Glu 195 200 205 Lys Thr Met Ala Glu Leu Thr Gin Leu Phe Asn Asp Met Lys Glu Leu 210 215 220 Val lie Glu Gin Gin Glu Asn Val Asp Val lie Asp Lys Asn Val Glu 225 230 235 240 Asp Ala Gin Gin Asp Val Glu Gin Gly Val Gly His Thr ABn Lys Ala 245 250 255 Val Lys Ser Ala Arg LyB Ala Arg Lys ABn Lys He Arg Cys Leu lie 260 265 270 lie Cys Phe lie lie Phe Ala lie Val Val Val Val Val Val Val Pro 275 2B0 285 Ser Val Val Glu Thr Arg Lys 290 295 </div>

Claims

<div class="application article clearfix printTableText" id="claims"> 41 256 42 5 WHAT WE CLAIM IS:

1. A<n isolated DNA sequence of a seel suppressor gene SSO, being selected from SSOl end SS02 sequences depicted in SEQ ID NO: 1 and SEQ ID NO: 3, respectively, and homologues thereof, which DNA sequence, when overexpressed in eukaryotic cells, renders said cells capable to produce increased amounts of secreted proteins.

2. A DNA sequence according to claim 1, wherein the sequence is derived from a fungus, and when overexpressed in a fungal host, renders said host capablc to produce increased amounts of secreted proteins.

3. A DNA sequence according to claim 2 being selected from SSOl and SS02 sequences coding for a polypeptide comprising essentially the amino acid sequence given in SEQ ID NO: 2 and SEQ ID NO: 4, respectively, or a functional fragment thereof.

4. A vector comprising a DNA sequence according to any one of the claims 1 to 3.

5. A vector according to claim 4, which vector is capable of replicating autonomously when transformed into eukaryotic cells.

6. A vector according to claim 4, which vector is capable of integrating into the chromosome when transformed into eukaryotic cells.

7. A vector according to claim 4 being a yeast expression vector wherein said DNA sequence is expressed under yeast gene regulatory regions.

8. A vector according to claim 7, wherein said yeast gene regulatory regions are selected from the group consisting of the promoter regions of the SSOl gene, SS02 2 3 HAY 1993 fWC*!V£t) 42 25 6 425 gene, SEC1 gene, GAL1 - GAL10 genes, the alcohol dehydrogenase gene ADH1, asparagine synthetase gene, and functional parts thereof.

9. A vector according to claim 4 being a filamentous fungus expression vector wherein said DNA sequence is expressed under regulatory regions functional in filamentous fungi.

10. A vector according to claim 9, wherein said regulatory regions functional in filamentous fungi are selected from the group consisting of the promoter regions of the sso, cbhl, cbh2, eg!1, eg!2, tefl, pf.k, pki, gpd, glucoamylase, a-amylase and alcohol dehydrogenase genes.

11. A vector according to claim 4 which is a fungal vector selected from the group consisting of YEpSSOl and YEpSS02, structures of which are given in Fig. 1A and IB, respectively.

12. Recombinant eukaryotic cells carrying a DNA sequence according to any one of claims 1 to 3 and expressing enhanced levels of Sso protein(s).

13. Recombinant eukaryotic cells according to claim 12 being fungal cells belonging to a species selected from the group consisting of Saccharomyces spp., Trichoderma spp., Kluyveromyces spp., Schizosaccharomyces pombe, Pichia spp., Hansenula spp., Yarrowia spp., Aspergillus spp. and Neurospora spp.

14. Recombinant eukaryotic cells according to claim 13 being fungal cells belonging to a species selected from Saccharomyces and Trichoderma.

15. Recombinant eukaryotic cells according to claim 14 being fungal cells of Saccharomyces cerevisiae strain VTT-C-92072 which has the deposition accession number DSM 7253. N .Z. A rfc'N r 2 3 - 43 256 425

16. Recombinant eukaryotic cells according to claim 14 being fungal cells of Saccharomyces cerevisiae strain VTT-C-92073 which has the deposition accession number DSM 7254.

17. A method for constructing new eukaryotic cells capable, of expressing enhanced levels of Sso protein(s), which method comprises: (a) isolating DNA sequence(s) selected from SSOl and SS02 sequences \ depicted in SEQ ID NO: 1 and SEQ ID NO: 3, respectively, and homologues thereof, coding for Sso protein(s), from a suitable donor organism; (b) constructing a vector carrying at least one of said DNA sequences; and (c) transforming at least one of the vectors obtained to suitable host cells.

18. A method according to claim 17, wherein the host to be transformed is selected from the group consisting of Saccharomyces spp., Trichoderma spp., Kluyveromyces spp., Schizosaccharomyces pombe, Pichia spp., Hansenula spp., Yarrowia spp., Aspergillus spp. and Neurospora spp.

19. A method according to claim 18, wherein said host belongs to a species selected from Saccharomyces and Trichoderma.

20. A process for producing increased amounts of secreted foreign or endogenous protein(s), by overexpressing the SSO gene(s), which process comprises: (a) isolating DNA sequence(s) coding for said protein(s) from a suitable donor organism; (b) constructing a vector carrying at least one of said DNA sequences; (c) transforming the vector obtained into a suitable host comprising DNA sequence(s) selected from SSOl and SS02 sequences depicted in SEQ ID NO: 1 and SEQ ID NO: 3, respectively, and homologues thereof, and expressing enhanced levels of Sso protein(s), to obtain recombinant host cells; or, alternatively, transforming the vector to a suitable host and retransforming this transformant with DNA seauenc?(g) fif.1f.ctfH from SSOl N.Z. PATENT Of"FIGS 2 3 MAY 1996 ftSCEiVED 44 256 425 and SS02 sequences depicted in SEQ ID NO: 1 and SEQ ID NO: 3, respectively, and homologues thereof, and screening for cells with enhanced production of said protein(s); and (d) cultivating said recombinant host cells under conditions permitting 5 expression of said protein(s).

21. A process for producing increased amounts of secreted foreign or endogenous protein(s), by overexpressing gene(s) interacting with the SSO gene, e.g. SECl, in the presence of nonnal or increased amounts of the Sso protein(s), which 10 process comprises: (a) isolating DNA sequencc(s) coding for said protein(s) from suitable donor organism; (b) constructing a vector carrying at least one of the said DNA sequences; (c) transforming the vector obtained into a suitable host expressing normal or 15 enhanced levels of Sso protein(s) and overexpressing other gene(s) interacting with SSO gene, e.g. SECl, to obtain recombinant host cells; or, alternatively, transforming the vector to a suitable host and retransforming this transformant with SSO or a gene homologous to SSO and by the gene(s) interacting with SSO gene, e.g. SECl and screening for cells with enhanced 20 production of said protein(s); and (d) cultivating said recombinant host cells under conditions permitting expression of said protein(s).

22. A process for increased production of an endogenous secreted protein, the 25 process comprising: (a) transforming cells producing said protein with DNA sequence(s) selected from SSOl and SS02 sequences depicted in SEQ ED NO: 1 and SEQ ID NO: 3, respectively, and homologues thereof, alone or together with gene(s) interacting with said SSO gene, such as SECl, 30 (b) screening for transfonnants producing enhanced level of said protein thus obtaining recombinant cells for enhanced protein production, and 45 256 42 5 (c) cultivating said recombinant cells in conditions permitting expression of said protein.

23. A process for efficient biomass production on a raw material or efficient hydrolysis of a raw material, which process comprises: (a) isolating DNA sequence(s) coding for endogenous or foreign hydrolytic enzyme(s) from a suitable donor organism; (b) constructing a fungal vector carrying at least one of the said DNA sequences; (c) transforming the vector obtained to a suitable fungal host comprising DNA sequence(s) selected from SSOl and SS02 sequences depicted in SEQ ID NO: 1 and SEQ ID NO: 3, respectively, and homologues thereof, and expressing enhanced levels of Sso protein(s), to obtain recombinant host cells, or, alternatively, transforming the vector to a suitable host and retransforming this transformant with DNA sequence(s) selected from SSOl and SS02 sequences depicted in SEQ ID NO: 1 and SEQ ID NO: 3, respectively, and homologues thereof, and screening for cells with enhanced production of said enzyme(s); and (d) cultivating said recombinant host cells under conditions permitting expression of said hydrolytic enzyme(s).

24. A process for efficient biomass production on a raw material or efficient hydrolysis of a raw material, by overexpressing genes interacting with the SSO gene, e.g. SECl, in the presence of nonnal or increased amounts of the Sso protein(s), which process comprises: (a) isolating the DNA sequence(s) coding for endogenous or foreign hydrolytic enzyme(s) from a suitable donor organism; (b) constructing a vector carrying at least one of said DNA sequences; (c) transforming the vector obtained to a suitable host expressing enhanced levels of proteins interacting with the Sso protein(s) in the presence of normal or increased amounts of the Sso protein(s) to ~P i ' ■»' M.Z. P sfCNT Off toe 2 3 MAY 1996 ■■II ■IIM—> 46 256425 obtain recombinant host cells, or, alternatively, transforming the vector to a suitable host and retransforming this transformant with SSO gene or a gene homologous to SSO and with the gene(s) interacting with SSO gene, such as SECl, and screening for cells with enhanced production of said enzyme(s); and (d) cultivating said recombinant host cells under conditions permitting expression of said hydrolytic enzyme(s).

25. An isolated DNA sequence as claimed in claim 1, substantially as herein described.

26. An isolated DNA sequence as claimed in claim 1, substantially as herein described and with reference to any one of the examples or figures.

27. A method as claimed in claim 17, substantially as herein described.

28. A method as claimed in claim 17, substantially as herein described with reference to any one of the examples or figures.

29. A process as claimed in any one of claims 20-24, substantially as herein described.

30. A process as claimed in any one of claims 20-24, substantially as herein described and with reference to any one of the examples or figures. </div>